A primary assumption that guides current approaches to in utero hematopoietic cellular transplantation (IUHCT) is that the early-gestation fetus has an immature immune system that is incapable of rejecting a donor cell transplant. For this reason, transplanting the cells prior to the maturation of the adaptive immune system leads to recognition of the donor cells as self rather than foreign. However, repeated failures in clinical cases of IUHCT that do not involve an immunodeficiency disease force a re-examination of this model of the fetal immune system. A survey of both clinical and laboratory reports of IUHCT reveal enhanced success when the immunodeficient host is also natural killer (NK) cell deficient. Preliminary studies in mice reveal that NK cells act as an early immune barrier to IUHCT. In order to establish NK cell tolerance and ensure successful engraftment, a minimum level of hematopoietic chimerism must be achieved prior to NK cell maturation. Below this chimerism threshold, the host NK cells predictably reject the graft. In this way, the chimerism threshold offers a logical explanation for past failures of clinical IUHCT. Fortunately, the identification of the chimerism threshold greatly facilitates study of the mechanisms linking the chimerism level to the emergence of NK cell tolerance following IUHCT. This concept drives the central hypothesis that the chimerism threshold provides the minimum amount of donor ligand recognition necessary for the selection of friendly NK cell phenotypes and repression of hostile ones. To challenge this hypothesis, the experiment in this proposal will determine the impact of the chimerism threshold on alloreactive NK cell: 1) selection; 2) cytotoxicity; and 3) memory. The expected outcome for the experiments in this proposal is the delineation of the mechanisms linking the chimerism level to the emergence of NK cell tolerance following IUHCT. This knowledge will have a dramatic impact on the field of IUHCT as it establishes a new paradigm for the early fetal immune response to allotransplantation. This directly affects the development of strategies to enhance the success of IUHCT such as donor cell dose, donor selection, booster transplants and therapeutic targeting of the host immune response. On a broader scale, these findings will guide further mechanistic study of fetal NK cell education.